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FIG. 5.

Differentiated 3T3-L1 adipocytes are sensitive to CyP-D–dependent necrotic cell death. A: 3T3-L1 fibroblasts (Fb) and differentiated 3T3-L1 adipocytes (Ad) were treated with 1 μmol/L STS for the time indicated.
The formation of proteolytic processed caspase 3 was detected by immunoblotting with a cleaved caspase 3–specific antibody.
B: Cell extracts were prepared from 3T3-L1 fibroblasts and at different times after induction of 3T3-L1 adipogenesis. The induction
of CyP-D protein was determined by immunoblotting and compared with the induction of the aP2 protein and actin as a loading
control. C: Cell extracts were prepared from 3T3-L1 Fbs and after 4 (D4) and 8 (D8) days of adipocyte differentiation. The extracts
were immunoblotted for the VDAC, COX-IV, and the p115 protein as a loading control. D: Cell extracts were prepared from differentiated 3T3-L1 adipocytes that were infected with a control lentivirus (MISSION
control vector, two representative cell lines) and from cells infected with shRNA directed against CyP-D (clones of 638 and
710, two representative cell lines). Total cell extracts were immunoblotted for CyP-D and β-actin as a loading control. E: Control and CyP-D–knockdown (KD) cells were differentiated for 8 days and incubated for 24 h in the absence of serum. The
cells were then treated with and without H2O2 (30 mmol/L) or ION (2 μmol/L) for 4 h. Cell extracts were prepared and immunoblotted for the presence of HMGB1 released into
the cell medium. F: Control and CyP-D–knockdown cells were differentiated for 8 days and incubated with and without and H2O2 (30 mmol/L) or ION (2 μmol/L) for 4 h in the presence of 10% FCS. Cell extracts were prepared and immunoblotted for CyP-D
and for the presence of HMGB1 released into the cell medium.